The cytochrome P450 (CYP) family of enzymes is a diverse group of enzymes most of which catalyse the oxidation of organic substances, including metabolic intermediates and toxins such as drugs. CYPs catalyse oxidation reactions via electron transfer from NADPH by a reductase, usually NADPH-cytochrome P450 reductase.
CYPs are the main enzymes involved in drug metabolism and bioactivation. They have thus found use in enzyme prodrug therapy, a tumour therapy aimed at reducing the systemic side-effects of antitumour medication. Medication is administered as a noncytotoxic prodrug and converted to its active form by drug-metabolising enzymes which are targeted to the tumour cells. Generally, tumour cells are transfected with a gene encoding the enzyme which is capable of bioactivating the inactive prodrug, followed by treatment of the patient with the prodrug (gene-directed enzyme prodrug therapy or GDEPT). Viral vectors are often used for transgene introduction, a strategy known as virus-directed enzyme prodrug therapy (VDEPT). This strategy can increase both the specificity and sensitivity of drug treatment, thus reducing side effects and improving efficacy.
CYP2B6 metabolises a range of toxic substances, including nicotine and the anticancer drugs cyclophosphamide, ifosfamide and thiotepa. Because of this activity, CYP2B6 has been used in models of VDEPT using the chemotherapeutic agent cyclophosphamide (CPA), which requires activation by CYP2B6 in order to render it cytotoxic. In patients treated with CPA in the standard way, activation by CYP2B6 occurs in the liver, and the active drug is then transported to the tumour site via the blood stream. Such non-specific administration can cause serious side effects due to cytotoxic activity on non-tumour cells, including cardiotoxicity, renal toxicity, bone marrow suppression and neurotoxicity. CYP2B6 is thus an ideal candidate for VDEPT, and has been successfully used in in vivo models of VDEPT using cytotoxicity assays (Waxman et al, Drug Metab Rev 1999, 31: 503-522; Tychopoulos et al, Cancer Gene Ther 2005, 12: 497-508).
One of the disadvantages of using CYP2B6 in a VDEPT strategy is the relatively low affinity of CYP2B6 for CPA, which shows a high Km. Modification of the CYP2B6 enzyme to increase its catalytic efficiency (Vmax/Km) for 4-hydroxylation of CPA has therefore been attempted, in order to improve the therapeutic effect of CYPB26 when used in VDEPT. The inventors have previously produced a double active site mutant (I114V/V477W) by mutagenesis of the active site of CYPB26 which had a four-fold increase in CPA-4-hydroxlation efficiency compared to the wild-type enzyme, mainly as a result of an increase in enzyme affinity (Nguyen et al, Mol Pharmacol 2008, 73: 1122-1133).
Another possibility for improving the efficiency of CYP2B6-mediated VDEPT is to co-transfect tumour cells with NADPH cytochrome P450 reductase (RED) in order to supply CYP2B6 with electrons, as basal cellular reductase activity may be insufficient and may thus be a limiting factor for CYP2B6 activity. Earlier work by the inventors has shown that supplying external RED in this way can increase CYP2B6-mediated toxicity. Two approaches were successfully used to supplement intratumoral RED activity and increase CYP2B6 activity: co-transfection of separate RED and CYB2B6 proteins, and creation of a CYP2B6-RED fusion protein which has both 4-hydroxylase activity and reductase activity (Tychopoulos et al, Cancer Gene Ther 2005, 12: 497-508).
These studies have shown that there is scope for improving the efficiency of CYP2B6 when used in enzyme-directed prodrug therapy. Such improvement could permit known drugs to be used on new tumour targets, as well as improving the response of known targets to drug therapy. Modulation of CYP2B6 activity is thus of great potential clinical importance and represents a useful potential tool in treating cancer.